APOBEC3A can activate the DNA damage response and cause cell-cycle arrest

Abstract

Human apolipoprotein-B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) proteins constitute a family of cytidine deaminases that mediate restriction of retroviruses, endogenous retro-elements and DNA viruses. It is well established that these enzymes are potent mutators of viral DNA, but it is unclear whether their editing activity is a threat to the integrity of the cellular genome. We show that expression of APOBEC3A can lead to induction of DNA breaks and activation of damage responses in a deaminase-dependent manner. Consistent with these observations, APOBEC3A expression induces cell-cycle arrest. These results indicate that cellular DNA is vulnerable to APOBEC3 activity and deregulated expression of APOBEC3A could threaten genomic integrity.

Figure 1

APOBEC3A expression can induce DNA damage responses. (A) APOBEC3A induces phosphorylation of histone H2AX. U2OS cells left untreated (MOCK) or transfected with plasmids expressing haemagglutinin (HA)-tagged A3A, A3B, A3C and A3G or the pcDNA3.1 control plasmid were fixed 24 h post-transfection, stained with HA and γH2AX antibodies, and analysed by confocal microscopy. (A,D,E) Nuclei were stained with DAPI. Results are representative of three independent experiments. (B) Western blot detection of phosphorylated H2AX in cells transfected with A3A. U2OS cells were transfected with APOBEC3 expression plasmids or pcDNA3.1 as control. Cells were collected at 48 h and lysates were analysed by western blot using HA, H2AX and γH2AX antibodies. Cells irradiated with 10 Gy were used as a positive control. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Results are representative of three independent experiments. (C) Transfected U2OS cells express A3A at physiological levels. A3A in lysates from U2OS transfected with 100 ng of A3A plasmid was compared with levels of endogenous A3A expressed in human peripheral blood mononuclear cells (PBMCs) stimulated with interferon (IFN)α (1,000 U/ml) for 24 h. Cell lysates were analysed by western blotting using A3A antibodies, and GAPDH was used as a loading control. (D) Activation of DNA repair proteins by A3A. U2OS cells were transfected with an A3A expression vector or pcDNA3.1 as control. After 24 h, cells were fixed and stained with antibodies to 53BP1, phosphorylated Nijmegen breakage syndrome 1 (Nbs1, S343), replication protein A32 (RPA32) and phosphorylated RPA32 (S4,8), and analysed by confocal microscopy. (E) Phosphorylated H2AX is not detected in cells transfected with the inactive A3A mutants C106S and E72Q. U2OS cells were transfected with plasmids expressing wild-type A3A, the C106S or E72Q mutants or the pcDNA3.1 control plasmid. After 24 h, cells were fixed, stained with HA and γH2AX antibodies and analysed by confocal microscopy. (F) Analysis of γH2AX staining in cells transfected with A3A or the C106S and E72Q mutants. Transfected cells were scored for γH2AX staining. The graph shows the percentage of cells with pan-nuclear γH2AX staining, γH2AX foci and background staining. (G,H) Analysis of phosphorylated H2AX in stable HepaRG cells expressing inducible A3A or the C106S mutant. Cells were grown in the presence or absence of doxycycline (1 μg/ml) for 24 h. Fixed cells were analysed by confocal microscopy (G) and cells lysates were analysed by western blot (H). Results are representative of three independent experiments. APOBEC3, apolipoprotein-B mRNA-editing catalytic polypeptide-like 3; DAPI, 4,6-diamidino-2-phenylindole.

Figure 2

Expression of APOBEC3A leads to DNA breaks. (A) Detection of DNA breaks in cells transfected to express A3A. U2OS cells were transfected with plasmids expressing wild-type A3A, the C106S mutant or the pcDNA3.1 control plasmid. Cells were fixed at 24 h, labelled by TUNEL and subsequently incubated with γH2AX and haemagglutinin (HA) antibodies. Cells treated for 10 min with DNAse I (3 U/ml) are shown as a positive control for TUNEL staining. Nuclei were stained with DAPI. Images are representative of three independent experiments. (B) Induction of DNA damage by A3A is not a consequence of apoptosis. U2OS cells were transfected with an A3A expression vector or pcDNA3.1, fixed at 24 h, and labelled by TUNEL and subsequently incubated with HA and the cleaved form of caspase 3 (CC3) antibodies. As a positive control, cells were treated with 20-μM camptothecin (CAMPT) for 20 h to induce apoptosis. Images are representative of three independent experiments. APOBEC3, apolipoprotein-B mRNA-editing catalytic polypeptide-like 3; DAPI, 4,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end-labelling.

Figure 3

Uracil–DNA glycosylase activity is required for A3A-induced DNA damage. (A) The UNG inhibitor (UGI) prevents H2AX phosphorylation in cells transfected with A3A. U2OS cells stably expressing UGI or control cells were transfected with A3A. Cells were fixed after 24 h, stained with haemagglutinin (HA) and γH2AX antibodies and analysed by confocal microscopy. Nuclei were stained with DAPI. Images are representative of three independent experiments. (B) Analysis of γH2AX staining in cells stably expressing UGI transfected with A3A. Cells showing HA staining were scored for γH2AX staining. The graph shows the percentage of cells with pan-nuclear H2AX activation, γH2AX foci and background staining. (C) Western blot detection of phosphorylated H2AX in U2OS cells stably expressing UGI transfected with A3A. Cells were transfected with A3A or the pcDNA3.1 control plasmid, collected at 24 h and lysates were analysed by western blotting, using HA, H2AX and γH2AX antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Images are representative of two independent experiments. (D) Induction of DNA breaks by A3A requires UNG activity. U2OS cells were co-transfected with A3A or the pcDNA3.1 control plasmid together with a plasmid expressing UGI (pLPC-UGI) or the empty vector. Cells were fixed after 24 h and labelled by TUNEL and subsequently incubated with γH2AX and HA antibodies. Nuclei were stained with DAPI and cells were analysed by confocal microscopy. Images are representative of three independent experiments. DAPI, 4,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end-labelling; UNG, uracil–DNA glycosylase.

Figure 4

APOBEC3A expression induces cell-cycle arrest. (A) Stable HepaRG cells expressing A3A are blocked in S phase. Inducible cells expressing wild-type and mutant A3A (HepaRG–A3A and HepaRG–C106S) were grown in the presence or absence of doxycycline (DOX, 1 μg/ml) and analysed for cell-cycle progression at different time points by propidium iodide staining. Results are representative of three independent experiments. (B) Graph shows the percentage of cells in G1, S and G2 phase for each sample at 36 h postinduction. (C) HepaRG, HepaRG–A3A and HepaRG–C106S were grown in the presence or absence of doxycycline and analysed for cell-cycle progression 24 h postinduction, using BrdU staining. Results are representative of three independent experiments. APOBEC3, apolipoprotein-B mRNA-editing catalytic polypeptide-like 3; BrdU, bromodeoxyuridine; UNG, uracil–DNA glycosylase.